Relapsing Pediatric T-Cell Acute Lymphoblastic Leukemia Downregulates T-Cell Properties and Upregulates Cell Adhesion

Relapse is the main cause of death from pediatric acute precursor T-cell leukemia (T-ALL), but the mechanisms underlying relapse remain poorly understood. We performed an integrated longitudinal multi-level omics analysis of 12 T-ALL patients to reveal common relapse-driving processes. Using high-throughput NGS-based techniques we compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression and methylation levels at initial diagnosis, remission and relapse in primary patient material and in patient derived xenografts (PDX).

The PDX models largely recapitulated the clonal architecture of the primary leukemias. Aberrant expression of T-ALL transcription factors (TAL1, TAL2, LY1L, LMO2, TLX1, TLX 3, NKX2.4, NKX2.5) was preserved from initial presentation to relapse in all of the patients, as were most of the large CNA and frequent leukemia-specific SNVs and InDels. The known activating mutations in NT5C2 were acquired at relapse by 7/12 patients. RNA-seq analysis showed 40 transcripts to be either down- or upregulated at the time of relapse in ≥4 patients. Methylome analysis of approximately 25,700 promoters showed 141 (0.6%) to be significantly altered (∆ ß ≥0.2; McNemar0.05), of which 109 were recurrently hypomethylated and 32 hypermethylated at relapse.

Functional enrichment classification revealed that 12 of the 32 promoters that were hypermethylated in relapse were highly T-cell specific and regulate genes involved in lymphopoiesis (DNTT, CD3G, CD3D, PTCRA, RAG2, RHOH, SIT1), encoding T cell receptors (TRBV30, TRBJ2-1, TRBV2, TRBV28) and the T-Cell Leukemia Homeobox 3 gene. Similarly, ontology terms associated with T-cell signaling and hematopoiesis were overrepresented among genes whose RNA expression was downregulated at relapse in ≥3 patients (CD4, CD8, CD9, DNTT, IL4, PTCRA RAG1, RAG2, TRAV13-2, TRAV18 and THEMIS). These changes in RNA expression and promoter methylation were reflected by cell surface marker expression in primary patient derived cells obtained at the time of initial presentation and relapse.

When considering the genes with significantly relapse-specific hypomethylated promoters and overexpressed transcripts, we identified genes associated with homophilic cell adhesion via plasma membrane adhesion molecules (GO:0007156) and containing cadherin conserved site (enrichment score for expression: 4.98 and for hypomethylation 2.39). Moreover, three of the patients acquired at relapse missense mutations in several cadherin-like genes (CDH23, CELSR1, PCDHA1, PCDHA10, PCDHA6, PCDHAC1, PCDHAC2 ; enrichment score: 2.47), although further studies are required to determine the functional consequences of these SNVs. Interestingly, the Suppressor Of Cytokine Signaling 2 (SOCS2) mRNA was the most frequently recurrent overexpressed transcript at relapse in 5/12 patients. SOCS2 is a negative regulator of cytokine receptor signaling via the JAK/STAT pathway and its deregulation was previously identified as part of a stemness-related molecular signature signifying unfavorable outcome in acute myeloid and lymphoblastic leukemia (Vitali et. al, 2015).

In conclusion, the longitudinal analysis of this cohort of patients identified relapse-specific alterations with potential functional impact. We postulate that relapsed T-ALLs lose T-cell specific properties and gain properties that result in resistance against chemotherapy. The latter may be related to the ability of relapse cells to upregulate the expression of cell adhesion molecules such as cadherins, protocadherins and integrins thus putatively modulating the leukemia/stroma interaction.